Many gastrointestinal (Gl) peptides, neurotransmitters and bioactive lipids bind to G protein-coupled receptors (GPCRs) and induce cell migration and proliferation in their target cells. These processes play an essential role in the repair of many forms of injury to the mucosa of the Gl tract, including erosions, ulcers, inflammatory bowel disease, enteropathic infections, ischemia and radiation. The broad, long-term objective of this proposal is to elucidate the signal transduction pathways that mediate GPCR-induced migration and proliferation in intestinal epithelial cells. In addition to signaling via classic second messengers (e.g., Ca2+, DAG, cAMP), an emerging theme is that GPCR agonists also induce tyrosine phosphorylation of multiple proteins, including the non-receptor tyrosine kinase p125 focal adhesion kinase (FAK) and the adaptor proteins p130 Crk-associated substrate (CAS) and paxillin, which are implicated in cell migration and proliferation. GPCR stimulation also induces rapid epidermal growth factor receptor (EGFR) transactivation. During the past funding period, our studies established that GPCR agonists, including regulatory peptides and bioactive lipids induce tyrosine phosphorylation cascades involving FAK, Src, Pyk2 and EGFR in a variety of cells, including intestinal epithelial cells. These studies form the basis for further exploration of the mechanisms by which tyrosine phosphorylation events contribute to cell migration and proliferation in GPCR-stimulated cells described in the current grant proposal. Based on our previous findings, our central hypothesis is that tyrosine phosphorylation cascades play a critical role in GPCR-induced cellular migration and mitogenesis in intestinal epithelial cells. This proposal will pursue the following specific hypotheses and aims: Hypothesis I. GPCR agonists induce tyrosine and serine phosphorylation of focal adhesion tyrosine kinases implicated in the regulation of cell migration and cell cycle progression. Aims emanating from hypothesis I: 1) Characterize the regulation of novel phosphorylation sites of FAK in response to GPCR agonists and EGFR ligands in IEC-18 and T84 intestinal epithelial cells. 2) Define the role of FAK in mediating migration, DNA synthesis, proliferation and signal transduction in intestinal epithelial cells. Hypothesis II: EGFR transactivation contributes to transduce mitogenic signaling by GPCR agonists in intestinal epithelial cells Aims emanating from hypothesis II: 3) Characterize the signal transduction pathways that mediate GPCR-induced EGFR transactivation in intestinal epithelial cells and determine the role of this process in GPCR-induced mitogenesis in these cells 4) Define the signaling mechanism(s) by which EGFR transactivation contributes to GPCR-mediated mitogenesis in intestinal epithelial cells.